Revisiting Wolbachia detections: Old and new issues in Aedes aegypti mosquitoes and other insects

Abstract Wolbachia continue to be reported in species previously thought to lack them, particularly Aedes aegypti mosquitoes. The presence of Wolbachia in this arbovirus vector is considered important because releases of mosquitoes with transinfected Wolbachia are being used around the world to suppress pathogen transmission and these efforts depend on a lack of Wolbachia in natural populations of this species. We previously assessed papers reporting Wolbachia in natural populations of Ae. aegypti and found little evidence that seemed convincing. However, since our review, more and more papers are emerging on Wolbachia detections in this species. Our purpose here is to evaluate these papers within the context of criteria we previously established but also new criteria that include the absence of releases of transinfections within the local areas being sampled which has contaminated natural populations in at least one case where novel detections have been reported. We also address the broader issue of Wolbachia detection in other insects where similar issues may arise which can affect overall estimates of this endosymbiont more generally. We note continuing shortcomings in papers purporting to find natural Wolbachia in Ae. aegypti which are applicable to other insects as well.

. CI is an essential component of the Wolbachia incompatible insect technique (IIT) applied for suppressing native mosquito populations, where released male Wolbachia carriers mate with native females to eventually reduce the size of mosquito populations.This approach is often accompanied by an additional radiation dose applied to released mosquitoes to ensure that any females carrying Wolbachia released due to inaccurate sexing do not become established (Zheng et al., 2019).CI is also an essential component of the replacement technique where releases of males and females carrying Wolbachia can result in the replacement of the natural mosquito population with those carrying the target Wolbachia strain capable of suppressing pathogen transmission (Hoffmann et al., 2011).
For both the population suppression and replacement approaches to work, it is essential that the targeted mosquito populations do not carry Wolbachia strains that prevent the expression of CI.For this reason, samples of target populations are typically screened prior to the initiation of releases.Any detection of natural Wolbachia should be followed up by crossing experiments to establish patterns of cross incompatibility which can be particularly complex in mosquito species like Culex pipiens (Atyame et al., 2011;Duron et al., 2006).This might lead to practitioners selecting different strains of Wolbachia for releases in a specific target area.
Aedes aegypti mosquitoes have been targeted by both the replacement approach and the suppression approach (Consortium & Ching, 2021;Crawford et al., 2020;Hoffmann et al., 2011;Indriani et al., 2023;Nazni et al., 2019).This species is the main vector of dengue virus in tropical areas and in the past has been considered as lacking Wolbachia (Gloria-Soria et al., 2018).In addition, suppression releases have targeted Aedes albopictus (Zheng et al., 2019) which is often naturally infected by two Wolbachia strains, wAlbA and wAlbB, and is considered a poorer vector of dengue (Lambrechts et al., 2010) but not other viruses such as chikungunya (Vega-Rúa et al., 2014).As Wolbachia releases have expanded to new countries, researchers have become interested in screening local Aedes species for Wolbachia, focussing particularly on Ae. aegypti.
In a previous report on Wolbachia detections in Ae. aegypti (Ross, Callahan, et al., 2020), we identified 8 studies purporting to detect natural infections.Unfortunately, there are issues involved in accurate detection and characterization of natural Wolbachia in mosquitoes and other insects which requires follow up work to confirm an infection and characterize it phenotypically.Of these studies, only two established lab populations to confirm the infection in lab stocks (Balaji et al., 2019;Kulkarni et al., 2019).We found at least one case where the infection then could not be confirmed from those stocks (Kulkarni et al., 2019;Ross, Callahan, et al., 2020).The main reason for this note is to reiterate issues with Wolbachia detection as more and more papers continue to report Wolbachia infections in Ae. aegypti (Table 1) and other species.
We discuss potential explanations for false positive detections and highlight cases where detections likely reflect released transinfections rather than natural infections.

| CHALLENG E S IN NE W S TUD IE S
We have identified 26 studies purporting to detect natural Wolbachia in Ae. aegypti in field populations (Table 1) and two others involving laboratory experiments based on one of these natural infections (Balaji et al., 2021;Balaji & Prabagaran, 2022).Twenty-one of these studies specifically claimed to detect natural Wolbachia infections as opposed to DNA sequences.Recent Wolbachia survey studies often cite previous detections uncritically as justification for conducting their own study, or as being in support of their own results, but continue to ignore issues raised previously.A challenge is that molecular approaches for detecting Wolbachia and other endosymbionts have their limitations.Molecular detection is often focused on one approach such as 16S rRNA which may detect Wolbachia among a community of other bacteria (Rodpai et al., 2023).This approach is prone to contamination, particularly when pooled samples are used or when a lab undertakes work on other species which may have a high abundance of Wolbachia.It also cannot readily be used to quantify endosymbiont densities, given that 16S primers may preferentially amplify some groups which depends on factors like primer efficiency and copy number (Větrovský & Baldrian, 2013).
In studies where multiple molecular markers are employed, these tend to give inconsistent patterns of Wolbachia presence (e.g.wsp + 16S rRNA comparisons) with one approach performing better in one study but the reverse occurring in another study (Somia et al., 2023;Wong et al., 2020).The incidence of Wolbachia detected is often very low (e.g.3.3% in Taiwan, (Chao & Shih, 2023); 5% Yunnan, China, (Zhang et al., 2022), 7% in Gabon (Zouache et al., 2022)) or cannot be estimated due to the use of pooled data (Vinayagam et al., 2023).Low frequencies are not expected to occur in populations with stable, maternally transmitted Wolbachia, and may reflect environmental contamination unless there is a clear geographic pattern.The strains detected often match existing strains being released (Somia et al., 2023) or strains present in related species (Chao & Shih, 2023;Zhang et al., 2022).In one example from northeastern India, only wAlbB Wolbachia was detected in sympatric Ae. albopictus and Ae.aegypti (Vinayagam et al., 2023), whereas the former species is typically double infected with wAlbB and wAlbA (Yang et al., 2022).
On the other hand, multiple Wolbachia types have also purported to have been detected in some population samples.Aedes aegypti from Manila were considered infected by at least 4 different Wolbachia including strains related to those from Drosophila melanogaster, Culex quinquefasciatus and Brugia malayi, although some of these appeared to be rare based on read numbers (Muharromah et al., 2023).This is an unusually high diversity of Wolbachia given that interactions among Wolbachia strains based on host effects associated with the Wolbachia typically drive some Wolbachia out of populations as well documented in Drosophila (Kriesner et al., 2013).
The presence of multiple Wolbachia strains was supported by additional work using different locally developed primers for common markers (Reyes et al., 2024), with a novel low-density strain being detected.However, it is worth noting that all three molecular papers At minimum, we recommend that any molecular detections should be followed up by qPCR on individuals with Wolbachiaspecific primers (e.g.wsp, ftz) and host genes included as controls.
Hosts should also be accurately identified such as using COI or ITS2 barcodes.qPCR or digital PCR methods are important in quantifying levels of infection, although read depth has also been successfully used (Muharromah et al., 2023) Since our previous review, there remains a lack of attempts to set up laboratory lines of Ae. aegypti for detailed evaluations, unlike other systems such as Anopheles mosquitoes where the presence of natural Wolbachia was previously in doubt (Walker et al., 2021).
Establishing laboratory lines of natural Wolbachia strains in Ae. aegypti should be relatively simple given the high frequency of Wolbachia apparently present in many populations (Table 1) and the ease at which this species can be reared and tested in the laboratory.If a laboratory stock is available, it is possible to undertake additional experiments to confirm the impact of Wolbachia on CI and also confirm the mode of inheritance as being maternal (Ross, Callahan, et al., 2020).There are cases of Wolbachia DNA being incorporated into host nuclear DNA (Brelsfoard et al., 2014;Nikoh et al., 2008) which then leads to nuclear rather than maternal inheritance being exhibited by the markers.CI experiments can also test whether any Note that first 8 cases listed here were considered in Ross, Callahan, et al. (2020); the other cases are new studies.

TA B L E 1 (Continued)
there has been no further validation beyond molecular detection since the original study (Balaji et al., 2019).Given its close similarity to wAlbB in Ae. albopictus we would expect it to cause CI or at least influence crossing patterns with this strain, however our attempts to contact the authors to perform an independent evaluation and test crossing patterns with Ae. aegypti transinfections have been unsuccessful.
It is possible that the low detections of some Wolbachia strains represent interspecific interactions, notably between (uninfected) Ae. aegypti and (naturally infected) Ae. albopictus.Although there is some variation among populations, Ae. albopictus females are typically infected by both wAlbB and wAlbA, with males tending to have a lower infection rate of wAlbA (Yang et al., 2022).
Interspecific matings between male Ae.albopictus and female Ae. aegypti occur at a low frequency in nature (Bargielowski & Lounibos, 2014;Tripet et al., 2011) and could result in Wolbachia being detected in Ae. aegypti females even if the host does not transmit the Wolbachia.Previous mating experiments (Ross, Axford, et al., 2020) indicate that Wolbachia can be detected in uninfected females after mating with an infected male although this effect was evident in wMelPop and wMel (a supergroup A infection like wAlbA from Ae. albopictus) rather than in the wAlbB strain tested in that paper However, wAlbB infections can have substantial genomic variation (Martinez et al., 2022) that may influence their detectability through PCR.
It is also possible (particularly for larval samples) that detections represent Wolbachia from other parasites such as nematodes.This is acknowledged in some papers (Thongsripong et al., 2018;Zouache et al., 2022) and could account for low level detections of Wolbachia.
Detections can reflect extremely low levels of Wolbachia that can also be quite diverse, which would seem to suggest other organisms or contaminants, particularly in pooled data.For instance, RNA sequencing of pooled adult mosquito samples from Yunnan indicated a very low density of Wolbachia in all adult pools with RPM at 1/40th the level recorded for Ae.albopictus (Li et al., 2023), whereas qPCR screening indicates higher Wolbachia titres of transinfections in Ae.
aegypti (c.f.Yang et al. (2022) and Ross et al. (2023)).The Wolbachia diversity based on 16S rRNA sequencing was also incredibly high in contrast to Wolbachia from other mosquitoes (Li et al., 2023).
In addition, as releases aimed at replacement and suppression continue to expand in countries and around the world (Figure 1), there is an increasing risk of interpreting detected Wolbachia as being natural rather than being associated with a release stock.An example of this is Somia et al. ( 2023) who detect two "natural" infections of Wolbachia in Jeddah, Saudi Arabia.Wolbachia releases have taken place in Jeddah following a detailed characterization of wMelM and wAlbB Wolbachia strains for release (Ross et al., 2023) and preparatory work at sites (Endersby-Harshman et al., 2021;Pagendam et al., 2022).With two strains being released, it is not surprising that the authors identified two clades of Wolbachia in Jeddah.
The authors do not discuss this possibility although they have previously published experimental work on Wolbachia field samples that they acknowledge as coming from releases (Algamdi et al., 2023).
Other detections of natural Wolbachia have also occurred in release areas such as in Selangor, Malaysia (Roslan et al., 2024;Wong et al., 2020) where Wolbachia releases were started some time ago (Nazni et al., 2019).
As more Wolbachia genomes are introduced into Ae.aegypti for suppression or replacement (Liu et al., 2022;Sarwar et al., 2022), any new detections of Wolbachia should ideally be characterized at this detailed level rather than relying on MLST markers to define strains.While  S1.A list of studies detecting natural Wolbachia strains in Ae. aegypti (b) is provided in Table 1.
detected natural infection might interfere with replacement by a different Wolbachia or IIT based suppression.Lab stocks can be used to undertake further characterization of Wolbachia in hosts, such as through fluorescence in situ hybridization (FISH)(Czarnetzki & Tebbe, 2004).In fact, laboratory stocks are essential to assess the concerns often used as justification for molecular screening of Aedes species.Balaji and Prabagaran (2022) have now performed additional experiments involving the laboratory population established byBalaji et al. (2019) to further characterize its phenotypic effects including CI.They show that the purported strain wAegB does not cause detectable CI, has no significant effect on fitness and does not provide protection against three bacterial pathogens(Balaji & Prabagaran, 2022).While this laboratory population has been confirmed to be positive for Wolbachia though PCR(Balaji & Prabagaran, 2022) and 16S rRNA sequencing(Balaji et al., 2021), the MLST system has been useful in the past, we are now at the stage where multiple Wolbachia variants within the wAlbB and wMel strains are being developed and released, reinforcing the usefulness of more F I G U R E 1 Maps of Wolbachia transinfection releases in Aedes aegypti (a) and detections of natural Wolbachia strains in Aedes aegypti (b).Data for transinfection releases were collated from published studies, press releases and personal communications.A list of sources for transinfection releases (a) is provided in Table Detections of purportedly natural Wolbachia strains in Aedes aegypti mosquitoes.a TA B L E 1 . Where Wolbachia levels are par- ination.However, the majority of studies listed in Table1that claim to have detected a natural Wolbachia infection do not acknowledge potential sources of contamination (17/21) or do not specify the need for further validation of these natural infections (15/21).